Developing maps of the protein-protein interaction network in human cells is crucial to understanding cancers and other human diseases that alter the regulatory state of normal cells. These maps seek to represent the individual protein-protein interactions that in combination lead to enzymatic regulation, cellular localization, or pathway facilitation. Global analysis of protein-protein interaction requires techniques, which query the interaction of a single protein with all of the proteins in the cell. Whether based on in vitro or in vivo assays, analysis is currently limited by the monoplex nature of most available technology. In this application, Trellis proposes to develop a highly multiplexed pangenomic library screen for performing library x library interaction assays. The screening technology adapts Trellis' microscopic multihued beads into multiplexed detectors of protein-protein interaction. Applied to filter lifts from high-density phage plaque replicas, the beads can provide a 100 x 10,000 interaction environment for quantitative analysis by a specialized reader, with potential to grow to 1,000 x 1,000,000. In preliminary studies, the company has demonstrated efficient and specific fluorescent bead binding to protein spotted on PVDF membrane filters. The size of the beads is selected to flow through the membrane without trapping and to bind with rapid kinetics. In addition, the company has demonstrated its ability to produce multiple levels of fluor staining for these nanobeads and to distinguish between bead populations by microscopic spectral analysis with an instrument suitable for conversion to an affordable plate reader. In Phase I, Trellis will (1) optimize reagents and protocols for fluorescent bead binding to membrane filters, (2) adapt an affordable commercial microscope platform to support spectral analysis of beads binding to phage plaque replicas, and (3) develop the first screening reagents for multiplexed detection of expressed proteins and use them to define protocols that can be applied on a large scale in Phase II. In addition to development of protein-protein network maps, the multiplex library screening technology will have application in development of recombinant antibodies, investigations of autoimmune disease and cancers, and analysis of peptide recognition motifs.